Hospital infections exact an enormous medical and economic toll, killing more people than AIDS, breast cancer, and auto accidents combined and costing the US healthcare system tens of billions of dollars annually. The emergence of pathogens resistant to all classes of antibiotics and the paucity of new antibiotics threaten to create vulnerability to infection reminiscent of the pre-antibiotic era. Rapid and accurate diagnostic testing enables timely implementation of the correct antimicrobial therapy which saves lives, decreases the severity of illness, improves antibiotic stewardship, and reduces healthcare costs. However, most testing today depends on slow 19th century microbiological culture methods which require days to get clinicians the answers they need to optimally treat their patients. Nucleic acid tests can be rapid but they are expensive and cannot reliably determine which antibiotics to use to treat most pathogens. Nucleic acid tests are limited by the often extensive genetic polymorphism of resistant bacteria due to their rapidly evolving genomes and facile inter-species gene transfer. We aim to develop the novel MultiPath platform to fill the technology gap, providing rapid, accurate, quantitative, and inexpensive identification and rapid antimicrobial susceptibility for panels of pathogens that cause the most important types of hospital infections. Because the method tests for growth in the presence of antibiotics, this rapid approach is robust to and unhindered by the genetic complexity of resistant pathogens. The simple and cost-effective digital imaging technology tests patient samples directly and eliminates sample preparation, cell-disruption, pipetting, biochemical purification, enzymes, and wash steps. Here we pro- pose to develop the first MultiPath panel tests for one of the most common hospital acquired infections, urinary tract infections, which can cause life-threatening blood infections. The project develops assays and consumable cartridges for a two stage UTI diagnostic approach that direct analyzes urine samples. First, the MultiPath UTI ID test will quantitatively detect 9 of the most common UTI pathogens in a rapid (15 min) and sensitive (<500 CFU/ml) assay. Then, if there is an infection, the pathogen in the patient's urine sample will be analyzed for susceptibility to a panel of relevant antibiotics in a rapid (3 hr) MultiPath UTI AST test. Specifically, we propose to (1) develop UTI target-specific MultiPath immunoreagents, (2) develop and demonstrate the multiplexed MultiPath UTI ID assay, (3) develop and demonstrate the performance of the MultiPath UTI ID cartridge, (4) develop and demonstrate the multiplexed MultiPath UTI AST assay, and (5) develop and demonstrate the MultiPath UTI AST cartridge. The successful project will deliver manufactured cartridge consumables with integrated stabilized reagents with performance data demonstrating the tests have the potential to meet rigorous regulatory specifications. Future work will refine and scale the consumable manufacturing processes and integrate the MultiPath UTI consumables with the high-throughput, on-demand, random-access, automated benchtop analyzer being developed with funding from other sources.